Glaucoma is an optic neuropathy characterized by selective retinal ganglion cell (RGC) degeneration with associated excavation of the optic nerve head, and one of the leading causes of blindness in the world. RGC death mechanisms have been shown to involve apoptosis. Examination of the optic nerve head can reveal signs of RGC axon loss, but wide variability exists and identification of disease is challenging. Characteristic field defects can confirm the diagnosis, but as many as 30%-50% of RGCs may be lost before defects are detectable by standard visual field testing. Thus, better advanced diagnostics are urgently needed, especially in high risk populations, which include older age, family history of glaucoma, black race, use of systemic or topical steroids, and high intraocular pressure. Furthermore, slowing disease progression and preserving quality of life are the main goals of glaucoma treatment. Indeed, several emerging therapeutic options for glaucoma, e.g., dietary vitamin B3 (niacin, a pre-cursor of NAD+) and overexpression of nicotinamide nucleotide adenylyltransferase 1 (Nmnat1), would benefit from a quantitative imaging biomarker. However, there exist no quantitative measures of RGC health to determine therapeutic effectiveness and better methods to monitor therapy are needed. Previously, we designed and synthesized peptide-based imaging agents that can penetrate cells via non- receptor-mediated endocytic pathways, gaining access to the cytosolic compartment. These cell-penetrating optical imaging agents contain quenched fluorophores flanking target protease sequences that are cleaved and activated by caspase-3, one key ?effector? protease in cells committed to apoptosis. Upon cleavage, these agents show caspase-3-dependent fluorescence signal amplification, thereby enabling high quality enzyme- specific single-cell imaging of apoptosis in vivo. We discovered that our cell-penetrating peptides preferentially accumulate in RGCs, the predominate cells of the retina selectively engaging endocytic pathways, and importantly, selectively injured in glaucoma. Because these RGCs are particularly accessible through an intravitreal approach, a routine ophthalmological procedure now performed everyday in the clinic, we are developing these cell-type-specific peptides for use in advanced diagnostics and therapeutic monitoring (not for screening the general population). This renewal application is focused on translation of this strategy through quantitative pre-clinical testing in advanced glaucoma models to monitor disease progression and quantify dietary (niacin) and gene therapy (Nmnat1) interventions, as well as toxicology analysis, and metabolite profiling. We will advance a lead peptide toward the clinic through a unique statistically-robust non-human primate model of glaucoma. These activities benefit from the combination expertise of this team in chemistry, molecular imaging, biochemistry, advanced animal models and vision biology.